Purified heat shock protein complexes

ABSTRACT

A method for purifying heat shock protein complexes is provided which comprises the steps of adding a solution containing heat shock protein complexes, in which heat shock proteins are associated with peptides, polypeptides, denatured proteins or antigens, to a column containing an ADP matrix to bind the heat shock, proteins complexes to the ADP matrix and adding a buffer containing ADP to the column to remove the heat shock protein complexes in an elution product. Additionally a method for synthesizing heat shock protein complexes and purifying the complexes so produced is provided which comprises the steps of adding heat shock proteins to an ADP matrix column to bind them to the matrix, adding a solution of peptides, polypeptides, denatured proteins or antigens to the column to bind them to the heat shock proteins as heat shock protein complexes and adding a buffer containing ADP to the column to remove the complexes in an elution product.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates generally to methods for purifyingand synthesizing heat shock protein complexes.

[0003] 2. Description of the Prior Art

[0004] Heat shock proteins (HSPs) are associated in cells with a broadspectrum of peptides, polypeptides, denatured proteins and antigens withwhich they form complexes. Such HSP-peptide complexes have beendescribed as being useful in vaccines against cancers and infectiousdiseases by Srivastava et al., “Heat shock protein-peptide complexes incancer immunotherapy” in Current Opinion in Immunology (1994),6:728-732; Srivastava, “Peptide-Binding Heat Shock Proteins in theEndoplasmic Reticulum” in Advances in Cancer Research (1993),62:153-177. The HSP-peptide complexes appear to work as vaccines,because they may function as antigen carrying and presentationmolecules. The development of vaccines using such antigens has beendescribed by Baltz, “Vaccines in the treatment of Cancer” in Am. J.Health-Syst. Pharm. (1995), 52:2574-2585. The antigenicity of heat shockproteins appears to derive not from the heat shock protein itself, butfrom the associated peptides, see Udono et al., “Heat Shock Protein70-associated Peptides Elicit Specific Cancer Immunity” in J. Exp. Med.(1993), 178:1391-1396; Srivastava et al., “Heat shock proteins transferpeptides during antigen processing and CTL priming” in Immunogenetics(1994), 39:93-98; Srivastava, “A Critical Contemplation on the Roles ofHeat Shock Proteins in Transfer of Antigenic Peptides During AntigenPresentation” in Behring Inst. Mitt. (1994), 94:37-47. HSPs appear to bepart of the process by which peptides are transported to the MajorHistocompatibility Complex (MHC) molecules for surface presentation.

[0005] A number of different HSPs have been shown to exhibitimmunogenicity including: gp96, hsp90 and hsp70, see Udono et al.,supra. and Udono et al., “Comparison of Tumor-Specific Immunogenicitiesof Stress-Induced Proteins gp96, hsp90, and hsp 70” in Journal ofImmunology (1994), 5398-5403; gp96 and grp94, Li et al., “Tumorrejection antigen gp96/grp94 is an ATPase: implications for proteinfolding and antigen presentation” in The EMBO Journal, Vol. 12, No. 8(1993), 3143-3151; and gp96, hsp90 and hsp70, Blachere et al., “HeatShock Protein Vaccines Against Cancer” in Journal Of Immunotherapy(1993), 14:352-356.

[0006] Heat shock proteins have been purified using a procedureemploying DE52 ion-exchange chromatography followed by affinitychromatography on ATP-agarose, see Welch et al., “Rapid Purification ofMammalian 70,000-Dalton Stress Proteins: Affinity of the Proteins forNucleotides” in Molecular and Cellular Biology (June 1985), 1229-1237.However, previous methods of purifying HSPs such as this one purify theheat shock proteins without the associated peptides. Other methods thatdo purify HSPs together with their associated peptides are complicatedand expensive.

SUMMARY OF THE INVENTION

[0007] Therefore, it is an object of the invention to provide a simpleand inexpensive method for purifying heat shock proteins together withtheir associated peptides, polypeptides, denatured proteins or antigensfrom cell lysates.

[0008] It is a further object of the invention to provide a method forsynthesizing heat shock protein complexes that is capable of formingthese complexes from heat shock proteins and peptides, polypeptides,denatured proteins or antigens from different cells and from differentspecies.

[0009] The present invention provides a method for purifying heat shockprotein complexes comprising the steps of adding a solution containingheat shock protein complexes, in which heat shock proteins areassociated with peptides, polypeptides, denatured proteins or antigens,to a column containing an ADP matrix to bind the heat shock proteinscomplexes to the ADP matrix and then adding a buffer containing ADP tothe column remove the heat shock protein complexes in an elutionproduct.

[0010] The present invention also provides a method for synthesizingheat shock protein complexes and purifying the complexes so produced byadding heat shock proteins to an ADP matrix column to bind them to thematrix, adding a solution of peptides, polypeptides, denatured proteinsor antigens to the column to bind them to the heat shock proteins asheat shock protein complexes and then adding a buffer containing ADP tothe column to remove the complexes in an elution product.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 is a drawing of a western blot of fractions taken from apurification using the ADP purification matrix;

[0012]FIG. 2 is a plot of HPLC data of material treated with NaCl afterbeing purified by the method of the invention and filtered through a20,000 molecular weight cut-off filter; and

[0013]FIG. 3 is a plot of HPLC data of material treated with ATP afterbeing purified by the method of the invention and filtered through a20,000 molecular weight cut-off filter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0014] In one preferred embodiment, the present invention provides amethod for isolating heat shock protein complexes from solutioncontaining heat shock proteins using an ADP matrix. Each of the heatshock protein complexes consists of a heat shock protein (HSP) that isbound tightly to an incomplete protein in a cell.

[0015] According to the method of the invention, solutions containingthese HSP complexes are added to a conventional column, such as anagarose gel column, to which ADP has been added to form an ADP matrix.Suitable ADP-agarose columns include those described in U.S. Pat. Nos.5,114,852; 5,268,465; 5132,407; and 5,541,095, the entire contents anddisclosures of which are hereby incorporated by reference. ADP has astrong affinity for the HSP complexes and unlike ATP, does not breakdown the HSP complexes when it binds to them.

[0016] Typically the solution from which the heat shock proteincomplexes are purified is a cell lysate from a tumor in which the HSPsare already present. However, the invention contemplates that thesolution containing HSP complexes to be purified may be produced bymixing an already purified heat shock protein with a cell lysate, amembrane isolate (materials isolated from a cell membrane) or a proteasetreated cell lysate containing peptides, polypeptides, denaturedproteins to produce a solution of HSP complexes. For the purposes of thepresent invention the terms “peptides” refers to all peptides andpolypeptides including denatured proteins, and recombinant or otherwisepurified tumor or infectious disease antigens that may be associatedwith heat shock proteins, either naturally or synthetically.

[0017] In order to increase the number of heat shock proteins in thesolution added to the ADP matrix column, the solution may be incubatedat a temperature of 37 to 50° C. and additional ADP may be added to thesolution prior to adding it to the column. If the HSP complex solutionis a cell lysate, additional HSPs may be added to the lysate to formadditional complexes.

[0018] A buffer solution containing ADP is added to the column to elutethe HSP complexes from the ADP matrix as an elution product containingthe HSP complexes. In addition to ADP, this buffer solution may inaddition contain small amounts of components such as sodium chloridethat aid in the removal of the complexes from the ADP matrix.

[0019] In order to produce a more purified elution product, after theHSP complexes have been bound to the ADP matrix, a purifying buffersolution may be added to the column to elute other proteins looselybound to the matrix. This purifying buffer solution preferably containsGTP or another non-adenosine containing nucleotide

[0020] The method of the invention takes advantages of the fact thatHSPs are associated with peptides inside the cell. This purificationmethod maintains the HSP-peptide association necessary to developvaccines or immunotherapeutic tools for tumors and for infectiousdiseases since HSPs have not been shown to be helpful as antigenswithout the associated peptides.

[0021] In another embodiment the invention provides a method forsynthesizing HSP complexes and purifying the complexes so produced. Inthis method, purified HSPs are bound to an ADP matrix column. Then apreparation of peptides, polypeptides, denatured proteins and/orantigens is added to an ADP matrix column to bind to the HSPs in thematrix. The method then proceeds similarly to the first embodiment ofthe invention. A buffer solution containing ADP is added to the columnto elute the HSP complexes from the ADP matrix as an elution productcontaining the HSP complexes. This buffer solution may contain smallamounts of components such as sodium chloride that aid in the removal ofthe complexes from the ADP matrix.

[0022] As with the first embodiment of the invention, a purifying buffersolution containing GTP or another non-adenosine containing nucleotidemay be added to the column to elute other proteins loosely bound to thematrix.

[0023] This second embodiment permits HSP complexes to be formed fromHSPs and peptides, denatured proteins or antigens from different cellsor even different species.

[0024] Although there are many heat shock proteins that may be used inthe method of the present invention, heat shock proteins that haveproven particularly useful include members of the hsp60 family, hsp70family, hsp90 family and the hsp104-105 family.

[0025] Members of the hsp60 family include hsp60, hsp65, rubisco bindingprotein, and TCP-1 in eukaryotes; and GroEl/GroES in prokaryotes; Mif4,and TCP1alpha and beta in yeast.

[0026] Members of the hsp70 family include DnaK proteins fromprokaryotes, Ssa, Ssb, and Ssc from yeast, hsp70, Grp75 and Grp78(Bip)from eukaryotes. FIG. 1 is a drawing of a western blot of fractionstaken from a purification using the method of the invention. The elutionwas started at fraction #10 and hsp70 protein appears in fraction #14.

[0027] Members of the hsp90 family include hsp90, g96 and grp94.

[0028] Members of the hsp104-170 family include hsp105 and hsp110.

[0029] The HSP/peptide complexes are eluted from the matrix using an ADPcontaining buffer. It also helps HSPs to be added to peptide mixturesand the complexes for use as a vaccine.

[0030] The invention will now be described by way of example. Thefollowing examples are illustrative and are not meant to limit the scopeof the invention which is set forth by the appointed claims.

EXAMPLE 1

[0031] A confluent T-75 of B16-F1 mouse melanoma cells was rinsed 3×with PBS. 1 ml of PBS was added and the cells were scraped to create asuspension. The suspension was spun for 5 minutes at 1000 rpm to pelletthe cells. The supernatant was removed and the cells resuspended in 1.5ml of a hypotonic buffer (30 mM NaHCO₃, pH 7.1). The suspension wastransferred to a glass tube and the cells were lysed with a Teflon®pestle and power drill. The lysate was transferred to a microcentifugetube and spun at high speed to pellet the undissolved fraction. Totalprotein of the lysate was determined using the Bradford method. Solutioncontaining 100 μg of total protein was brought up to 300 μl total volumewith the addition of Phosphate buffer (0.1M KH₂PO₄, 10 mM NaCl, 1 mMEDTA, pH 7.2) and the solution was added to a 5 ml ADP-agaraose column(linked through C-8, Sigma Chemical Co.) and allowed to run into thecolumn with 5 ml of Phosphate buffer and then buffer B (20 mM TRIS, 20mM NaCl, 15 mM EDTA, 15 mM Beta-mercaptoethanol, pH 7.5) with 60 mM ADPwas added at the start of fraction 10 to elute the complexes. Aftercompletion of the run, 50 μl of each fraction was run onto a 7.5% SDSPAGE gel, transferred to nitrocellulose, probed with an antibody for theinducible and constitutive hsp70 (N27, Stressgen Biotechologies), andthen a secondary alkaline phosphate linked antibody. A blot wasdeveloped in a buffer containing BCIP and NBT. A drawing of this plot isshown in FIG. 1.

EXAMPLE 2

[0032] PC-3 lysate was run over a agarose column containing an ADPmatrix according the method of the invention. The ASP containingfraction was then eluted with ADP. The eluted fraction containing HSPswas filtered using a 20,000 molecular weight cut-off (MWC) filter andseveral rinses of buffer A (25 mMTris, 20 mM Hepes, 47.5 mM KCl, and2.25 mM Mg(OAc)2, pH 7.2) were applied. The sample was split into twomicrocentrifuge tubes and either ATP (to 10 mM) or NaCl (to 1 mM) wasadded. The tubes were then incubated overnight at 37° C. Each solutionwas then spun through a 20,000MWC filter and the filtered material wasapplied to an HPLC column. The HPLC was accomplished using a C18 reversephase column (Vydac, 201TB54). The starting buffer was 0.1% TFA in dH₂Oand the material was eluted using a gradient of 0.1% TFA in ACN. FIG. 2shows HPLC data for the material treated with NaCl after being purifiedwith the ADP matrix and filtered through the 20,000 molecular weightcut-off filter. FIG. 3 shows the HPLC data for the material treated withATP after being purified with the ADP matrix and filtered through the20,000 molecular weight cut-off filter. The HPLC data in FIGS. 2 and 3is consistent with the data for hsp70 described in Udono et al., “HeatShock Protein 70-associated Peptides Elicit Specific Cancer Immunity” inJ. Exp. Med. (1993), 178:1391-1396.

[0033] Although the present invention has been fully described inconjunction with the preferred embodiment thereof with reference to theaccompanying drawings, it is to be understood that various changes andmodifications may be apparent to those skilled in the art. Such changesand modifications are to be understood as included within the scope ofthe present invention as defined by the appended claims, unless theydepart therefrom.

What is claimed:
 1. A method for purifying heat shock protein complexescomprising the steps of: adding a heat shock protein having at least oneof the group consisting of peptides, polypeptides, denatured proteinsand antigens associated therewith to a column containing an ADP matrixto bind the heat shock proteins complexes to the ADP matrix; and addinga buffer containing ADP to the column to remove the heat shock proteincomplexes in an elution product.
 2. The method of claim 1 furthercomprising the step of adding a purifying buffer solution to the columnto elute proteins that do not bind with the ADP matrix.
 3. The method ofclaim 1 wherein the solution containing heat shock protein complexescomprises a cell lysate.
 4. The method of claim 1 further comprising thestep of incubating the solution containing heat shock protein complexesat a temperature of 37 to 50° C. prior to adding the solution to thecolumn to induce heat shock proteins present in the solution to bind topeptides, polypeptides, denatured proteins and antigens present in thesolution to form heat shock protein complexes.
 5. The method of claim 1further comprising the step of adding ADP to the solution containingheat shock protein complexes prior to adding the solution to the columnto induce heat shock proteins present in the solution to bind topeptides, polypeptides, denatured proteins and antigens present in thesolution to form heat shock protein complexes.
 6. The method of claim 1further comprising the step of adding a buffer solution containing GTPto the column to elute proteins other than heat shock proteins that areloosely bound to the matrix.
 7. The method of claim 1 further comprisingadding purified heat shock proteins to the solution containing heatshock proteins prior to adding the solution to the column.
 8. The methodof claim 1 wherein the heat shock protein complexes include complexes inwhich the heat shock protein comprises one of the group consisting ofhsp60, hsp65, rubisco binding protein and TCP-1 from eukaryotes;GroEL/GroES, Mif4, TCPalpha and TCPbeta from yeast.
 9. The method ofclaim 1 wherein the heat shock protein complexes include complexes inwhich the heat shock protein comprises of one of the group consisting ofhsp104, hsp105 and hsp110.
 10. The method of claim 1 wherein the heatshock protein complexes include complexes in which the heat shockprotein comprises one of the group consisting DnaK proteins fromprokaryotes; Ssa, Ssb, and Ssc from yeast; hsp70, Grp75 and Grp78(Bip)from eukaryotes.
 11. The method of claim 1 wherein the heat shockprotein complexes include complexes in which the heat shock proteincomprises one of the group consisting of hsp90, g96 and grp94.
 12. Themethod of claim 1 further comprising the step of producing the heatshock protein complex by mixing a heat shock protein with a complexingagent selected from the group consisting of peptides, polypeptides,denatured proteins and antigens.
 13. A method for synthesizing heatshock protein complexes comprising the steps of: adding a heat shockprotein to a column containing an ADP matrix to bind the heat shockprotein to the ADP matrix; adding a complexing solution comprising acomplexing agent selected from the group consisting of peptides,polypeptides, denatured proteins and antigens to the column to form heatshock protein complexes with the heat shock protein bound to the ADPmatrix; and adding a buffer containing ADP to the column remove the heatshock protein complexes in an elution product.
 14. The method of claim13 further comprising the step of adding a purifying buffer solution tothe column to elute proteins that do not bind with the ADP matrix. 15.The method of claim 13 wherein the complexing solution comprises apeptide mixture selected from the group consisting of cell lysates,membrane isolates, and protease treated cell lysates.
 16. The method ofclaim 13 further comprising the step of incubating the solutioncontaining heat shock protein complexes at a temperature of 37 to 50° C.prior to adding the solution to the column to induce heat shock proteinspresent in the solution to bind to peptides, polypeptides, denaturedproteins and antigens present in the solution to form heat shock proteincomplexes.
 17. The method of claim 13 further comprising the step ofadding ADP to the solution containing heat shock protein complexes priorto adding the solution to the column to induce heat shock proteinspresent in the solution to bind to peptides, polypeptides, denaturedproteins and antigens present in the solution to form heat shock proteincomplexes.
 18. The method of claim 13 further comprising the step ofadding a buffer solution containing GTP to the column to elute proteinsother than heat shock proteins that are loosely bound to the matrix. 19.The method of claim 13 further comprising adding purified heat shockproteins to the solution containing heat shock proteins prior to addingthe solution to the column.
 20. The method of claim 13 wherein the heatshock protein complexes include complexes in which the heat shockprotein comprises one of the group consisting of hsp60, hsp 65, rubiscobinding protein and TCP-1 from eukaryuotes; GroEL/GroES, Mif4, TCPalphaand TCPbeta from yeast.
 21. The method of claim 13 wherein the heatshock protein complexes include complexes in which the heat shockprotein comprises of one of the group consisting of hsp104, hsp105 andhsp110.
 22. The method of claim 13 wherein the heat shock proteincomplexes include complexes in which the heat shock protein comprisesone of the group consisting DnaK proteins from prokaryotes; Ssa, Ssb,and Ssc from yeast; hsp70, Grp75 and Grp78(Bip) from eukaryotes.
 23. Themethod of claim 13 wherein the heat shock protein complexes includecomplexes in which the heat shock protein comprises one of the groupconsisting of hsp90, g96 and grp94.